WO2023048236A1 - Peptide - Google Patents

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Publication number
WO2023048236A1
WO2023048236A1 PCT/JP2022/035392 JP2022035392W WO2023048236A1 WO 2023048236 A1 WO2023048236 A1 WO 2023048236A1 JP 2022035392 W JP2022035392 W JP 2022035392W WO 2023048236 A1 WO2023048236 A1 WO 2023048236A1
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Prior art keywords
group
peptide
asp
phe
ala
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PCT/JP2022/035392
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English (en)
Japanese (ja)
Inventor
王明 柏木
隆 岡添
光介 相川
信介 山東
淳平 森本
晃司 門田
愛 木幡
Original Assignee
Ａｇｃ株式会社
国立大学法人東京大学
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Application filed by Ａｇｃ株式会社, 国立大学法人東京大学 filed Critical Ａｇｃ株式会社
Priority to EP22872987.7A priority Critical patent/EP4406961A1/fr
Priority to CN202280061928.3A priority patent/CN117999272A/zh
Priority to JP2023549749A priority patent/JPWO2023048236A1/ja
Publication of WO2023048236A1 publication Critical patent/WO2023048236A1/fr
Priority to US18/605,993 priority patent/US20240287131A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to peptides containing amino acid residues with fluorine atoms introduced into their side chains.
  • CPP cell penetrating peptides
  • Typical CPPs include peptides derived from the TAT protein of HIV virus (Patent Document 1) and poly-Arg sequence peptides (Patent Document 2). This can be combined with a medicinal peptide to transport the medicinal peptide into cells (eg, Patent Document 3, Non-Patent Document 1).
  • Non-Patent Document 2 Fluorine-containing amino acids and peptides containing them are expected to be used in the pharmaceutical field as physiologically active substances.
  • Non-Patent Document 4 Compounds with a polyfluoro structure are known to be stable in vivo, have low toxicity, and excel in being taken up into cells and escaping from endosomes. Utilizing this property, it has been reported that a peptide dendrimer using lysine in which the side chain amino group is perfluoroacylated as a constituent amino acid can be used for gene delivery (Non-Patent Document 5). However, since it is a dendrimer, it cannot form a hybrid in which it is bound to a therapeutically active peptide, a nucleic acid, or a protein that serves as an antibody drug, unlike CPP.
  • An object of the present invention is to provide a peptide containing an amino acid residue with a fluorine atom introduced into its side chain and a method for producing the same.
  • a peptide in which two or more amino acids are peptide-bonded, At least one side chain of the amino acid residue constituting the peptide has the following general formula (1)
  • Z 1 is a linking group other than a divalent, trivalent, or tetravalent alkylene group;
  • Rf is a C 1-30 alkyl group substituted with at least two fluorine atoms (the C 1-30 alkyl When the group has 2 or more carbon atoms, it may have 1 to 5 etheric oxygen atoms between the carbon atoms), -SF 5 , or -SF 4 -CR 101 R 102 -CR 103 R 104 Cl (R 101 , R 102 , R 103 and R 104 are each independently a hydrogen atom, a fluorine atom or a chlorine atom, but R 101 , R 102 , R 103 and R 104 are fluorine atoms); n3 is 1, 2, or 3, and a black circle means a bond] is a peptide.
  • the Rf is the following general formula (f-1) or (f-2)
  • Rf P is a fully halogenated C 1-10 alkyl group containing at least 2 or more fluorine atoms (the C 1-10 alkyl group is, when the carbon atoms are 2 or more, ether may have a bonding oxygen atom), n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, a black circle means a bond]
  • Z 1 is represented by the following general formula (2)
  • Z 2 is a linking group other than a divalent, trivalent, or tetravalent alkylene group; Rh is a hydrogen atom or a C 1-6 alkyl group, and a black circle means a bond]
  • the amino acid residue in which the group represented by the general formula (1) is a side chain is an amino acid residue in which 1 to 3 of the Rf are directly or indirectly linked to the side chain of a natural amino acid.
  • the peptide according to any one of [1] to [9], wherein the C-terminus or N-terminus may be protected with a protecting group.
  • the following general formula (11) the following general formula (11)
  • Rh is a hydrogen atom or a C 1-6 alkyl group ; an alkyl group or a benzyl group; X is a 9-fluorenylmethyloxycarbonyl group or a tert-butoxycarbonyl group; Z is a C 1-6 alkoxy group, a hydroxyl group, or an amino group]
  • the peptide of [1] or [2], which is a tripeptide represented by [12] The peptide of any one of [1] to [11], which is cell membrane permeable.
  • the peptide according to the present invention has excellent cell membrane permeability because fluorine atoms are introduced into the side chains. Therefore, the peptide is expected to be used in the pharmaceutical field as a physiologically active substance.
  • FIG. 4 shows the results of flow cytometry of HeLa cells treated at 37° C. for 1 hour in a sample solution containing (diethylamide of fluorescent substance Alexa Fluoro 647) (FD-1).
  • FIG. 2 shows the results of measurement of mean fluorescence intensity (MFI) of HeLa cells treated at 37° C. for 1 hour in a sample solution containing (FD-1).
  • MFI mean fluorescence intensity
  • peptide fluorescent conjugate 1 Alexa-Ala-Asp(C 8 F 17 )-Phe-NH 2 ) (PFCJ-1)
  • peptide fluorescent conjugate 2 Alexa-Ala-Asp(bis-C 4 F 9 )-Phe-NH 2 )
  • fluorescent dye 1 diethylamide of fluorescent substance Alexa Fluoro 647) (FD1) or fluorescent conjugate of cell membrane-permeable peptide Cys-TAT (47-57) (TAT -Alexa), showing the results of flow cytometry of HeLa cells treated at 37°C for 1 hour.
  • FIG. 4 shows the results of measurement of mean fluorescence intensity (MFI) of HeLa cells treated at 37° C. for 1 hour in a sample solution containing a gate (TAT-Alexa).
  • peptide fluorescent conjugate 1 Alexa-Ala-Asp(C 8 F 17 )-Phe-NH 2 ) (PFCJ-1)
  • peptide fluorescent conjugate 3 Alexa-Ala- Mean fluorescence intensity ( MFI ) is a diagram showing the results of measurement.
  • peptide fluorescent conjugate 1 Alexa-Ala-Asp (C 8 F 17 )-Phe-NH 2 ) (PFCJ-1)
  • peptide fluorescent conjugate 3 Alexa-Ala-Asp Mean fluorescence intensity (MFI) of HeLa cells treated for 1 hour at 37° C.
  • peptide fluorescent conjugate 1 Alexa-Ala-Asp(C 8 F 17 )-Phe-NH 2 ) (PFCJ-1)
  • peptide fluorescent conjugate 2 Alexa-Ala-Asp(bis-C 4 F 9 )-Phe-NH 2 )
  • peptide fluorescent conjugate 3 Alexa-Ala-Asp(C 12 H 25 )-Phe-NH 2 ) (PFCJ-3)
  • peptide fluorescent conjugate 6 Alexa-Ala-Asp(C 4 F 9 )-Phe-NH 2 ) (PFCJ-6
  • peptide fluorescent conjugate 7 Alexa-Ala-Asp(C 6 F 13 )-Phe-NH 2 ) (PFCJ-7 )
  • peptide fluorescent conjugate 9 Alexa-Ala-Asp (SF 4
  • FIG. 4 shows the results of flow cytometry of HeLa cells treated at 37° C. for 1 hour in a sample solution containing (diethylamide of fluorescent substance Alexa Fluoro 647) (FD-1).
  • FIG. 2 shows the results of measurement of mean fluorescence intensity (MFI) of HeLa cells treated at 37° C. for 1 hour in a sample solution containing (FD-1).
  • MFI mean fluorescence intensity
  • peptide fluorescent conjugate 1 Alexa-Ala-Asp(C 8 F 17 )-Phe-NH 2 ) (PFCJ-1)
  • peptide fluorescent conjugate 12 Alexa-Ala-Asp(CH 2 CH 2 C 6 F 13 )-Phe-NH 2 ) (PFCJ-12) or in a sample solution containing fluorescent dye 1 (FD-1)
  • MFI mean fluorescence intensity
  • C p1-p2 (p1 and p2 are positive integers satisfying p1 ⁇ p2) means a group having p1 or more and p2 or less carbon atoms.
  • a "C 1-30 alkyl group” is an alkyl group having 1 to 30 carbon atoms, and may be linear or branched.
  • a “C 2-30 alkyl group” is an alkyl group having 2 to 30 carbon atoms, and may be linear or branched.
  • C 1-30 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert- pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, heneicosyl group , docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl
  • C 1-10 alkyl group is an alkyl group having 1 to 10 carbon atoms and may be linear or branched.
  • a “C 2-10 alkyl group” is an alkyl group having 2 to 10 carbon atoms, and may be linear or branched.
  • C 1-10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert- pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group and the like.
  • C 1-6 alkyl group is an alkyl group having 1 to 6 carbon atoms, and may be linear or branched.
  • Examples of C 1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert- A pentyl group, a hexyl group, and the like can be mentioned.
  • a "C 6-14 aryl group” is an aromatic hydrocarbon group having 6 to 14 carbon atoms, and a C 6-12 aryl group is particularly preferred.
  • Examples of the C 6-14 aryl group include phenyl group, naphthyl group, anthryl group, 9-fluorenyl group and the like, with phenyl group being particularly preferred.
  • the “optionally substituted C 6-14 aryl group” means one or more, preferably 1 to 1, hydrogen atoms bonded to the carbon atoms of the C 6-14 aryl group. Three are groups substituted with other functional groups. When having two or more substituents, the substituents may be the same or different.
  • the substituents include a nitro group, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), a C 1-6 alkyl group, a C 1-6 alkoxy group, and a methylenedioxy group (-O-CH 2 -O-) and the like.
  • optionally substituted C 6-14 aryl group examples include phenyl group, naphthyl group, anthryl group, 4-nitrophenyl group, 4-methoxyphenyl group, 2,4-dimethoxyphenyl group, 3, 4-dimethoxyphenyl group, 4-methylphenyl group, 2,6-dimethylphenyl group, 3-chlorophenyl group, 1,3-benzodioxol-5-yl group and the like.
  • C 6-14 aryl- C 1-6 alkyl group means that one hydrogen atom bonded to a carbon atom of the C 1-6 alkyl group is a C 6-14 aryl group is a group substituted with
  • the C 6-14 aryl group in the C 6-14 aryl-C 1-6 alkyl group can be exemplified by a phenyl group, a naphthyl group, an anthryl group, a 9-fluorenyl group and the like, and a phenyl group or a 9-fluorenyl group is particularly preferred. .
  • the C 1-6 alkyl group in the C 6-14 aryl-C 1-6 alkyl group is preferably a C 1-4 alkyl group.
  • Examples of C 6-14 aryl-C 1-6 alkyl groups include benzyl group, diphenylmethyl group, triphenylmethyl group, 2-phenylethyl group, 9-anthrylmethyl group, 9-fluorenylmethyl group and the like. is mentioned.
  • halogen atom means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
  • a "halogen atom other than a fluorine atom” means a chlorine atom, a bromine atom, or an iodine atom.
  • halogen atom other than fluorine atom a chlorine atom or a bromine atom is preferable, and a chlorine atom is particularly preferable.
  • C 1-6 alkoxy group refers to a group in which an oxygen atom is bonded to a C 1-6 alkyl group having 1 to 6 carbon atoms.
  • a C 1-6 alkoxy group may be straight or branched. Examples of C 1-6 alkoxy groups include methoxy, ethoxy, propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
  • an "etheric oxygen atom” is an oxygen atom that connects carbon atoms, and does not include an oxygen atom in which oxygen atoms are connected in series.
  • An alkyl group having Nc carbon atoms (Nc is an integer of 2 or more) can have up to Nc ⁇ 1 oxygen atoms having an ether bond.
  • compound n means a compound represented by formula (n).
  • the peptide according to the present invention is a peptide consisting of two or more amino acids, wherein at least one side chain of the amino acid residues constituting the peptide is a group represented by the following general formula (1).
  • a black circle means a bond.
  • an amino acid in which a group represented by the following general formula (1) is bonded to the ⁇ -carbon may be referred to as a "fluorine-containing amino acid”
  • at least one side of an amino acid residue constituting a peptide A peptide whose chain is a group represented by the following general formula (1) is sometimes referred to as a "fluorine-containing peptide”.
  • Rf is a C 1-30 alkyl group substituted with at least two fluorine atoms, —SF 5 , or —SF 4 —CR 101 R 102 —CR 103 R 104 Cl.
  • Rf is a C 1-30 alkyl group substituted with at least two fluorine atoms
  • the C 1-30 alkyl group has 2 or more carbon atoms (in the case of a C 2-30 alkyl group), It may have 1 to 5 etheric oxygen atoms between carbon atoms.
  • one or more hydrogen atoms bonded to carbon atoms may be further substituted with halogen atoms other than fluorine atoms.
  • the C 1-30 alkyl group for Rf is preferably a C 1-20 alkyl group, more preferably a C 1-10 alkyl group, still more preferably a C 2-10 alkyl group, and a C 2-8 alkyl group. Even more preferable.
  • the C1-30 alkyl group is a C2-30 alkyl group, it may have 1 to 5 etheric oxygen atoms between carbon atoms.
  • the number of hydrogen atoms substituted with fluorine atoms is not particularly limited as long as it is 2 or more, for example, preferably 3 or more, more preferably 6 or more, and further 7 or more. preferable.
  • Rf examples include trifluoromethyl, pentafluoroethyl, heptafluoropropyl, nonafluorobutyl, perfluoropentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluorononyl, perfluorodecyl, difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, 1,1,2,2,3,3-hexafluoropropyl group, 1,1,2,3,3,3-hexafluoropropyl group, 1,1,2,2,3,3-hexafluorohexyl group, 1,1,2,2,3,3-hexafluorooctyl group , 1,1,2,2,3,3-hexafluorodecyl group, 1,1,2,2,3,3-hexafluorooc
  • Rf is a group having 2 carbon atoms
  • Rf is a pentafluoroethyl group rather than a 1,1,1-trifluoroethyl group (CF 3 —CH 2 —)
  • Rf is preferably a linear group, and in the case of a branched group, 1,1,1,3,3,3-hexafluoropropane -2-yl group ((CF 3 ) 2 -CH-) and (CF 3 ) 2 -CF- group are preferred.
  • Rf When Rf is a group having 4 carbon atoms, Rf may be a linear group or a branched group. In the case of a branched group, it is preferably a group in which a hydrogen atom bonded to a carbon atom constituting the alkylene group portion is substituted with a fluorine atom, or a completely fluorinated group.
  • Rf is preferably a group represented by the following general formula (f-1) or (f-2).
  • Rf P represents a fully halogenated C 1-10 alkyl group containing at least 2 or more fluorine atoms.
  • Rf P is a group in which all hydrogen atoms in a C 1-10 alkyl group are substituted with halogen atoms, and at least two or more of these halogen atoms are fluorine atoms.
  • Rf P has 2 or more carbon atoms, that is, when it is a fully halogenated C 2-10 alkyl group, it may have 1 to 5 etheric oxygen atoms between carbon atoms.
  • two Rf P may be the same group or different groups.
  • n1 is an integer of 0-10 and n2 is an integer of 0-9.
  • n1 and n2 are 0, both represent single bonds. That is, when n1 is 0, the group represented by general formula (f-1) is Rf P —, and when n2 is 0, the group represented by general formula (f-2) is (Rf P ) 2 -CH-.
  • Rf P is a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluoro A hexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, wherein n1 is an integer of 0 to 4, and Rf P is a trifluoromethyl group, a pentafluoroethyl group, a hepta fluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl
  • Rf P is a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluoro A hexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, wherein n2 is an integer of 0 to 4, and Rf P is a trifluoromethyl group, a pentafluoroethyl group, a hepta fluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl
  • Rf examples include difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, 1,1,2,2,3,3 -hexafluoropropyl group, 1,1,2,3,3,3-hexafluoropropyl group and the like.
  • Rf is a group represented by —SF 4 —CR 101 R 102 —CR 103 R 104 Cl
  • R 101 , R 102 , R 103 and R 104 each independently represent a hydrogen atom, a fluorine atom, or a chlorine atom.
  • two or more of R 101 , R 102 , R 103 and R 104 are fluorine atoms.
  • Specific examples of the group represented by -SF 4 -CR 101 R 102 -CR 103 R 104 Cl include -SF 4 -CF 2 -CF 2 Cl, -SF 4 -CF 2 -CFCl 2 , -SF 4 -CF 2 -CHF-Cl, -SF 4 -CF 2 -CCl 3 , -SF 4 -CF 2 -CHCl 2 , -SF 4 -CF 2 -CH 2 Cl, -SF 4 -CFCl-CFCl 2 , - SF 4 -CFCl-CHF-Cl and -SF 4 -CHF-CHF-Cl are included.
  • Z 1 is a linking group other than a divalent, trivalent, or tetravalent alkylene group, and n3 is 1, 2, or 3.
  • Z 1 is not particularly limited as long as it is a divalent to tetravalent group other than an alkylene group.
  • the aryl group and the heteroaryl group the groups mentioned above can be used. However, groups consisting only of alkylene groups and groups in which the portion connecting to Rf is an alkylene group are excluded.
  • Z 1 is a divalent linking group
  • the group represented by general formula (1) includes one Rf group It becomes a group having When Z 1 has a trivalent nitrogen atom, the nitrogen atom is represented by the general formula (1) by binding two Rf groups directly or via another divalent linking group
  • a group can be a group with two Rf groups.
  • Z 1 may be a linking group having a group (cyclic group) in which a hydrogen atom is removed from the ring, or may be a linking group having no ring group.
  • the group represented by general formula (1) can be a group having 2 or 3 Rf groups.
  • the cyclic group includes a group obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, an aromatic ring, or a heterocyclic ring.
  • the heterocyclic ring is preferably a ring in which 1 to 3 carbon atoms of an aromatic ring are substituted with one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur atoms.
  • the cyclic group may be a group obtained by removing a hydrogen atom from a monocyclic ring, or a group obtained by removing a hydrogen atom from a condensed ring.
  • the cyclic group contained in Z 1 in general formula (1) is preferably a group obtained by removing 2 to 4 hydrogen atoms from cyclohexane, benzene, imidazole, or indole.
  • the ring group is a 1,4-phenylene group, a 1,3-phenylene group, a 1,5 -phenylene group or 1,3,5-substituted phenyl group, preferably 1,4-phenylene group or 1,3,5-substituted phenyl group.
  • a linking group obtained by combining any one of these groups with a C 1-6 alkylene group is also preferred.
  • Z 1 in the general formula (1) preferably has an oxygen atom at the linking portion with Rf
  • Z 1 in general formula (1) is preferably a linking group represented by general formula (2) below.
  • Z2 is a linking group other than a divalent, trivalent, or tetravalent alkylene group.
  • Z 2 is not particularly limited as long as it is a divalent to tetravalent group other than an alkylene group.
  • the aryl group and the heteroaryl group the groups mentioned above can be used. However, groups consisting only of
  • Z 2 in general formula (2) can be the same as those listed for Z 1 .
  • Rh is a hydrogen atom or a C 1-6 alkyl group.
  • Rh is preferably a C 1-3 alkyl group, more preferably a methyl group or an ethyl group.
  • Z 1 is a group represented by general formula (2)
  • Rf is represented by general formula (f-1) or (f-2).
  • a group that is a group can be mentioned.
  • the amino acid residue whose side chain is the group (-Z 1 -(Rf)n3) represented by the general formula (1) is directly or indirectly attached to the side chain of the natural amino acid.
  • Peptides that are amino acid residues with 1-3 Rf groups linked to are preferred.
  • the fluorine-containing peptide according to the present invention includes one or two hydrogen atoms of the side chain amino group of an arginine residue, an asparagine residue, a glutamine residue, or a lysine residue, and -Rf or —Z 3 —(Rf)n4 (Z 3 is a linking group other than a 2-, 3-, or 4-valent alkylene group, excluding groups whose linking portion to Rf is an alkylene group.
  • Z 3 is not particularly limited as long as it is a divalent to tetravalent group other than an alkylene group.
  • the groups mentioned above can be used. However, groups consisting only of alkylene groups and groups in which the portion connecting to Rf is an alkylene group are excluded.
  • Z3 the same linking group as Z1 and Z2 can be used.
  • the fluorine-containing peptides according to the present invention include peptides containing at least one amino acid residue whose side chain is -Z 1 -(Rf)n3. At least one side chain among amino acid residues constituting the peptide may be -Z 1 -(Rf)n3, and side chains of all amino acid residues may be -Z 1 -(Rf)n3.
  • the plurality of -Z 1 -(Rf)n3s may be the same as each other.
  • the amino acid residue having -Z 1 -(Rf)n3 as a side chain may be at the N-terminus, the C-terminus, or other than at the terminal.
  • the fluorine-containing peptide according to the present invention may be a peptide consisting of 2 or more amino acids, and a peptide consisting of 3 or more amino acids is also preferable.
  • the fluorine-containing peptide according to the present invention is preferably a peptide consisting of 2-40 amino acids, more preferably a peptide consisting of 3-20 amino acids.
  • the amino acid sequence of the fluorine-containing peptide according to the present invention is not particularly limited.
  • peptides with a higher proportion of hydrophobic (non-polar) side chains than polar side chains have better cell membrane permeability. Therefore, the fluorine-containing peptide according to the present invention can also have an amino acid sequence containing relatively many amino acid residues having hydrophobic side chains.
  • the fluorine-containing peptide according to the present invention when used by linking it to a hydrophobic substance, the fluorine-containing peptide has an amino acid sequence containing relatively many amino acid residues having polar side chains, so that the entire molecule Hydrophobicity and hydrophilicity can be balanced, and the cell membrane permeability can be further improved.
  • the N-terminus of the fluorine-containing peptide according to the present invention may be protected with an amino-protecting group.
  • the N-terminal protective group is not particularly limited as long as it is an amino group protective group, and for example, an amino group protective group used in peptide synthesis can be used.
  • protective groups for amino groups include tert-butoxycarbonyl (Boc) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group, benzyloxycarbonyl (Cbz) group, allyloxycarbonyl (Alloc) group, 2,2, Carbamate-based protecting groups such as 2-trichloroethoxycarbonyl (Troc) group can be mentioned.
  • a tert-butoxycarbonyl (Boc) group or a 9-fluorenylmethyloxycarbonyl (Fmoc) group is preferred because it can be deprotected under mild conditions.
  • the C-terminus of the fluorine-containing peptide according to the present invention may be protected with a protecting group.
  • the C-terminal-protecting group is not particularly limited as long as it is a carboxy-protecting group, and for example, a carboxy-protecting group used in peptide synthesis can be used.
  • Specific examples of the carboxy-protecting group include a group represented by the following general formula (p-1), a 2-(9,10-dioxo)anthrylmethyl group, a benzyloxymethyl group, and a phenacyl group. is the protecting group of choice.
  • R 3 is an optionally substituted C 6-14 aryl group
  • R 4 and R 5 are each independently a hydrogen atom or an optionally substituted C 6-14 aryl group.
  • a black circle means a bond.
  • Carboxy-protecting groups include benzyl, diphenylmethyl, triphenylmethyl, 4-nitrobenzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, 4- methylbenzyl group, 2,6-dimethylbenzyl group, 3-chlorobenzyl group, 9-anthrylmethyl group, piperonyl group, 2-(9,10-dioxo)anthrylmethyl group, benzyloxymethyl group, phenacyl group, etc. is mentioned.
  • the C-terminal carboxy-protecting group is preferably a benzyl group or a triphenylmethyl group, more preferably a benzyl group, in that deprotection can be performed under mild conditions.
  • the amino acid residue whose side chain is not -Z 1 -(Rf)n3 is not particularly limited, and may be an amino acid residue of an ⁇ -amino acid, It may be a ⁇ -amino acid residue, a ⁇ -amino acid residue, or a ⁇ -amino acid residue. Moreover, it may be an amino acid residue of an L-amino acid or an amino acid residue of a D-amino acid.
  • Amino acid residues whose side chain is not -Z 1 -(Rf)n3 contained in the fluorine-containing peptide according to the present invention include protein-constituting amino acids, D-forms thereof, and modified side chains thereof. It is preferably an amino acid residue of a modified amino acid.
  • Amino acids that make up proteins include glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine, proline, aspartic acid, glutamic acid, lysine, arginine, histidine, and the like.
  • modified amino acids in which protein-constituting amino acids are modified include, for example, hydrogen atoms of amino groups in the side chains of lysine, arginine, and histidine, which are the above-mentioned amino group-protecting groups and Pbf (N- ⁇ - Amino acids substituted with (2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl) groups; the hydrogen atoms of the carboxy groups in the side chains of aspartic acid and glutamic acid are the carboxy group-protecting groups listed above. or tert-butyl group substituted with an alkyl group; and amino acids substituted with a benzyl group for the hydrogen atom of the cysteine thiol group.
  • fluorine-containing peptides according to the present invention include tripeptides represented by the following general formula (11).
  • Rf and n3 are the same as Rf and n3 in general formula (1) above
  • Z2 and Rh are the same as Z2 and Rh in general formula (2) above. .
  • R 11 and R 12 are each independently a C 1-6 alkyl group or a benzyl group.
  • R 11 and R 12 are each independently preferably a methyl group or a benzyl group, and R 11 is preferably a methyl group and R 12 is a benzyl group. Especially preferred.
  • X is a 9-fluorenylmethyloxycarbonyl group (Fmoc) or a tert-butoxycarbonyl group (Boc).
  • Z is a C 1-6 alkoxy group, hydroxyl group or amino group. When Z is a C 1-6 alkoxy group, Z is particularly preferably a methoxy group.
  • the fluorine-containing peptide according to the present invention can be produced by a general peptide synthesis method, except that at least an amino acid having -Z 1 -(Rf)n3 as a side chain is used as a starting amino acid.
  • it can be carried out by a peptide solid-phase synthesis method.
  • a fluorine-containing peptide can be easily synthesized using an automatic peptide synthesizer using an amino acid having -Z 1 -(Rf)n3 as a side chain.
  • an amino acid having a side chain of -Z 1 -(Rf)n3 is preferred.
  • a peptide can be produced by sequentially condensing an amino acid whose C-terminus is bound to a solid phase with an amino acid whose amino group is protected, and releasing the peptide from the solid phase. It is preferable to use an amino acid raw material whose amino group is protected with a Boc group or an Fmoc group.
  • the side chain functional group of the amino acid starting material is preferably protected with a protecting group.
  • Protective groups for side chain functional groups include Boc group, triphenylmethyl group, benzyl group, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) group and the like.
  • Condensing agents that form peptide bonds include, for example, N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (WSC), benzotriazol-1-yloxy-trisdimethyl.
  • DCC N,N-dicyclohexylcarbodiimide
  • WSC 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide
  • benzotriazol-1-yloxy-trisdimethyl benzotriazol-1-yloxy-trisdimethyl.
  • BOP aminophosphonium hexafluorophosphate
  • pyBOP benzotriazol-1-yloxytrispyrrolidinophosphonium hexafluorophosphate
  • HBTU 2-(1H-benzotriazol-1-yl)-1,1,3, 3-tetramethyluronium hexafluorophosphate
  • 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate 1-cyano-2- ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU);
  • N-hydroxybenzotriazole (HOBt) ethyl (hydroxyimino)cyanoacetate (oxyma) and the condensing agent can be mixed in a preferable ratio and used.
  • a method of activating the carboxy terminus may be used to form a peptide bond, and examples of activating agents include N-hydroxysuccinimide, p-nitrophenyl ester, pentafluorophenyl ester, and the like.
  • Bases used for forming peptide bonds include, for example, triethylamine and diisopropylethylamine (DIPEA).
  • Solvents used in the peptide bond-forming reaction include, for example, chloroform, dichloromethane (DCM), dichloroethane (DCE), acetonitrile (MeCN), N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO) and the like.
  • the Boc group and Fmoc group which are protective groups for the amino-terminal amino group of peptides or amino acids, can be removed with trifluoroacetic acid (TFA) or piperidine, respectively.
  • TFA trifluoroacetic acid
  • Protective groups for side chain functional groups of amino acid residues of peptides can be removed by, for example, TFA, hydrogen fluoride (HF), trifluoromethanesulfonic acid, and the like.
  • TFA in the solid-phase peptide synthesis method, can be used as a method for removing peptides or peptides having protective groups attached to side chain functional groups of amino acid residues from the peptide solid-phase synthetic resin. Detachment of the peptide from the solid-phase peptide resin and detachment of the protective group of the side chain functional group of the amino acid residue can be carried out simultaneously in the same reaction system. Alternatively, each can be performed independently.
  • Peptide solid-phase synthetic resins for solid-phase peptide synthesis include, for example, 4-hydroxymethyl-3-methoxyphenoxybutyric acid-benzhydrylamine-polystyrene resin, p-benzyloxybenzyl alcohol-polystyrene resin, oxime resin, and the like, which are commercially available. can be used.
  • the target peptide or intermediate thereof is isolated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, normal phase chromatography, recrystallization, extraction, and fractional crystallization. be able to. Also, the peptides thus obtained can be converted into respective salts by conventional methods.
  • the protective group for the amino group or carboxyl group of the produced fluorine-containing peptide can be deprotected as necessary. Deprotection can be carried out by a conventional method depending on the type of protecting group.
  • the fluorine-containing peptide according to the present invention Since the fluoroalkyl group, —SF5, —SF 4 —CR 101 R 102 —CR 103 R 104 Cl contains many fluorine atoms, the fluorine-containing peptide according to the present invention has excellent cell membrane permeability. In addition, it is difficult to be degraded by peptidases because its structure is significantly different from that of natural peptides. Utilizing these properties, the fluorine-containing peptide according to the present invention is expected to be used as a physiologically active substance in the medical field. For example, the fluorine-containing peptide according to the present invention can be expected to be used as a DDS carrier that delivers medicinal ingredients to target cells.
  • the target cell of the functional ingredient is obtained.
  • the functional component may be a peptide, protein, or low-molecular-weight compound.
  • -Z 1 -(Rf)n3 for some of the side chains of the amino acid residues constituting the functional peptide exhibiting physiological activity to the extent that the function of the functional peptide is not impaired.
  • the functional peptide can improve cell membrane permeability and intracellular retention time.
  • Non-natural amino acids as described in International Publication No. 2021/002407 require stereoselective reaction or optical resolution, and it is very troublesome to synthesize various fluorine-containing amino acid derivatives. .
  • synthesis of peptides as described in WO2021/002408 derived from such non-natural amino acids is also very laborious.
  • the peptides of the present invention can be derived from natural amino acids, and sterically defined amino acids can be synthesized without optical resolution.
  • it is possible to synthesize a fluorine-containing unit by separately preparing it and reacting it with a natural amino acid, and various fluorine-containing amino acids can be synthesized in the same production process, which is advantageous in terms of cost.
  • the NMR apparatus used for the analysis of the examples and comparative examples was JNM-ECZ400S (400 MHz) manufactured by JEOL Ltd. tetramethylsilane was 0 PPM in 1 H NMR, and C 6 F 6 was -162 PPM in 19 F NMR. bottom.
  • Tris(dibenzylideneacetone)dipalladium (0) (10 mol%) and phosphine (20 mol%) were added to a solution of compound (2a) (173 mg, 0.2 mmol) in THF (2 mL) at 0°C and stirred, After adding phenylsilane (2 equivalents), the temperature was raised to room temperature and the mixture was stirred for 2 hours. The reaction mixture was then quenched with HCl (1N) (10 mL) and extracted twice with dichloromethane.
  • compound (9) was prepared using Dalton Trans. , 2012, vol. 41, p. 8368 and NewJ. Chem. , 2017, vol. 41, p. 7729, Electronic Supplementary Information, and synthesized according to the above reaction scheme.
  • Fmoc- A crude product of Asp(tris-t-C 4 F 9 )-OAll (compound (10a)) was obtained.
  • Fmoc-Asp(C 12 H 25 ) was prepared in the same manner as in Production Example 1 using 4-dodecylaniline (compound (11)) (78 mg, 0.3 mmol) instead of 4-(perfluorooctyl)aniline.
  • a crude product of -OAll (compound (12a)) was obtained.
  • Fmoc-Asp was prepared in the same manner as in Production Example 1 using 4-(perfluorohexyl)aniline (Compound (15)) (411 mg, 1.0 mmol) instead of 4-(perfluorooctyl)aniline.
  • a crude product of (C 6 F 13 )-OAll (compound (16a)) was obtained.
  • Fmoc-Asp was prepared in the same manner as in Production Example 1 using 4-(perfluorodecyl)aniline (Compound (15)) (611 mg, 1.0 mmol) instead of 4-(perfluorooctyl)aniline.
  • Compound (15) 611 mg, 1.0 mmol
  • 4-(perfluorooctyl)aniline 611 mg, 1.0 mmol
  • a crude product of (C 10 F 21 )-OAll (compound (21)) was obtained.
  • Rink-amide resin 39 mg, 0.025 ⁇ mol
  • DMF 2 mL
  • a 20% piperidine/DMF solution (2 mL) was then added to the tube, stirred for 3 minutes, and then washed with DMF (2 mL) three times.
  • the Fmoc group was deprotected by adding 20% piperidine/DMF solution (2 mL) again and stirring for 12 minutes.
  • Fmoc-Asp(C 8 F 17 )-OH (84 mg, 0.10 mmol, 4.0 equivalents) synthesized in Preparation Example 1 and Fmoc-Ala-OH (31 mg, 0.10 mmol, 4.0 equivalents) were treated in the same manner. equivalent).
  • Alexa Fluor registered trademark 647 manufactured by ThermoFisher Scientific was bound to the N-terminus of the synthesized tripeptide (H-Ala-Asp(C 8 F 17 )-Phe-NH 2 ) having a heptadecafluorooctyl group.
  • Alexa Fluor 647-NHS ester (0.2 mg) dissolved in dry DMSO (20 ⁇ L), H-Ala-Asp(C 8 F 17 )- dissolved in dry DMSO (200 ⁇ L) in a 1.5 mL black tube.
  • Phe-NH 2 (4.0 eq) and DIPEA (3.0 eq) dissolved in dry DMSO (10 ⁇ L) were added.
  • Conjugate 1 was obtained as a blue solid (33% yield calculated by fluorometer). Fluorescence was measured using a Nano Drop (registered trademark) spectrophotometer ND-1000 at an emission wavelength of 650 nm.
  • H-Ala-Asp(bis-C 4 F 9 )-Phe-NH 2 was bound to Alexa Fluor 647 in the same manner as in Example 1 to obtain H-Ala-Asp(bis-C 4 F 9 ).
  • the fluorescent conjugate 2 of -Phe-NH 2 was obtained as a blue solid (32% yield calculated by fluorometer based on dye).
  • Example 2 the same method as in Example 1 was performed using Fmoc-Asp(C 6 F 13 )-OH synthesized in Production Example 7 instead of Fmoc-Asp(C 8 F 17 )-OH as the amino acid to be condensed. to give H-Ala-Asp(C 6 F 13 )-Phe-NH 2 (2.1 mg, 2.8 ⁇ mol, 11% yield).
  • a tripeptide was synthesized in the same manner as in Example 1 using Fmoc-Asp(C 10 F 21 )-OH instead of Fmoc-Asp(C 8 F 17 )-OH as the amino acid to be condensed.
  • H-Ala-Asp(C 10 F 21 )-Phe-NH 2 (1.8 mg, 1.9 ⁇ mol, 8% yield).
  • Fmoc-Asp(C 10 F 21 )-OH was obtained by subjecting the compound (22a) synthesized in Production Example 7 to a deallylation reaction in the same manner as in Production Example 1. The resulting product was used for tripeptide synthesis without purification.
  • Fmoc-Asp(SF 4 CF 2 CF 2 Cl)-OH synthesized in Production Example 7 was used instead of Fmoc-Asp(C 8 F 17 )-OH, and the same procedure as in Example 1 was performed. to obtain H-Ala-Asp(SF 4 CF 2 CF 2 Cl)-Phe-NH 2 (1.8 mg, 2.7 ⁇ mol, yield 11%).
  • H-Ala-Asp(C 4 F 9 )-Phe-NH 2 was bound to Alexa Fluor 647 in the same manner as in Example 1 to form H-Ala-Asp(C 4 F 9 )-Phe-NH.
  • the fluorescent conjugate 6 of 2 was obtained as a blue solid (54% yield calculated on the fluorometer based on dye).
  • H-Ala-Asp(C 6 F 13 )-Phe-NH 2 was bound to Alexa Fluor 647 in the same manner as in Example 1 to form H-Ala-Asp(C 6 F 13 )-Phe-NH.
  • the fluorescent conjugate 7 of 2 was obtained as a blue solid (50% yield calculated on the fluorometer based on dye).
  • H-Ala-Asp(CH 2 CH 2 C 6 F 13 )-Phe-NH 2 was combined with Alexa Fluor 647 in the same manner as in Example 1 to form H-Ala-Asp(CH 2 CH 2 C
  • the fluorescent conjugate 12 of 6 F 13 )-Phe-NH 2 was obtained as a blue solid (96% yield calculated by fluorometer based on dye).
  • H-Ala-Asp(C 10 F 21 )-Phe-NH 2 was combined with Alexa Fluor 647 in the same manner as in Example 1 to form H-Ala-Asp(C 10 F 21 )-Phe.
  • the fluorescent conjugate 8 of -NH2 was obtained as a blue solid (72% yield calculated by fluorometer based on dye).
  • H-Ala-Asp(SF 4 CF 2 CF 2 Cl)-Phe-NH 2 was bound to Alexa Fluor 647 in the same manner as in Example 1 to obtain H-Ala-Asp(SF 4 CF 2
  • the fluorescent conjugate 9 of CF 2 Cl)-Phe-NH 2 was obtained as a blue solid (42% yield calculated by fluorometer based on dye).
  • H-Ala-Asp(tris-t-C 4 F 9 )-Phe-NH 2 was bound to Alexa Fluor 647 in the same manner as in Example 1, and H-Ala-Asp(tris-t
  • the fluorescent conjugate 10 of -C 4 F 9 )-Phe-NH 2 was obtained as a blue solid (89% yield calculated by fluorometer based on dye).
  • Example 2 The same procedure as in Example 1 was performed using Fmoc-Asp(CH 2 CH 2 C 8 F 17 )-OH synthesized in Production Example 8 instead of Fmoc-Asp(C 8 F 17 )-OH as the amino acid to be condensed.
  • the tripeptide was synthesized by the method to give H-Ala-Asp(CH 2 CH 2 C 8 F 17 )-Phe-NH 2 (5 mg, 6.27 ⁇ mol, 25% yield).
  • H-Ala-Asp(CH 2 CH 2 C 8 F 17 )-Phe-NH 2 was combined with Alexa Fluor 647 in the same manner as in Example 1 to form H-Ala-Asp(CH 2 CH 2 C
  • the fluorescent conjugate 11 of 8 F 17 )-Phe-NH 2 was obtained as a blue solid (80% yield calculated by fluorometer based on dye).
  • a tetrapeptide is synthesized in the same manner as in Example 1 using Fmoc-Asp(C 8 F 17 )-OH as the amino acid to be condensed, and H-Cys-Ala-Asp(C 8 F 17 )- Phe-NH 2 (16 mg, 17 ⁇ mol, 22% yield) was obtained.
  • Example 2 the same method as in Example 1 was performed using Fmoc-Asp(C 6 F 13 )-OH synthesized in Production Example 7 instead of Fmoc-Asp(C 8 F 17 )-OH as the amino acid to be condensed. to synthesize the tetrapeptide to give H-Cys-Ala-Asp(C 6 F 13 )-Phe-NH 2 (12 mg, 15 ⁇ mol, 57% yield).
  • H-Ala-Asp(C 12 H 25 )-Phe-NH 2 was bound to Alexa Fluor 647 in the same manner as in Example 1 to form H-Ala-Asp(C 12 H 25 )-Phe-NH.
  • the fluorescent conjugate 3 of 2 was obtained as a blue solid (23% yield calculated by fluorometer based on dye).
  • the combined organic phase was concentrated under reduced pressure, diluted with ethyl acetate, and washed with HCl (1N), saturated aqueous sodium bicarbonate, and saturated brine.
  • the washed organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product of Boc-RFAA(C8)-Phe-OMe.
  • the steric structure of RFAA (C8) contained was determined to be the (S) form by X-ray structural analysis.
  • Rigaku VariMax Dual Saturn was used for X-ray structure analysis.
  • the washed organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude Boc-Ala-[(R)-RFAA(C8)]-Phe-OMe.
  • the crude product was dissolved in DCM, then reprecipitated with hexane and filtered to give pure Boc-Ala-[(R)-RFAA(C8)]-Phe-OMe (0.20 mmol, yield 85.0 mmol). 4%) was obtained.
  • Boc-Ala-[(S)-RFAA(C8)]-Phe-OMe (diastereomer B) (45.7 ⁇ mol) was deprotected in the same manner to obtain H-Ala-[(S)-RFAA. (C8)]-Phe-OMe (diastereomer B) (21.1 ⁇ mol, 46.2% yield) was obtained.
  • the fluorescent substance Alexa Fluor647 was bound to the N-terminal of the synthesized deprotected tripeptide (H-Ala-[(R)-RFAA(C8)]-Phe-OMe).
  • Alexa Fluor 647-NHS ester (1.75 mg) dissolved in dry DMSO (175 ⁇ L), H-Ala-RFAA(C8)-Phe- dissolved in dry DMSO (50 ⁇ L) OMe (3.0 eq), DIPEA (3.0 eq) dissolved in dry DMSO (71 ⁇ L), and 124 ⁇ L dry DMSO were added.
  • the mixture was kept stirring overnight at room temperature.
  • H-Ala-[(S)-RFAA(C8)]-Phe-OMe (diastereomer B) (1.241 ⁇ mol) was similarly coupled with Alexa Fluor TM 647 to form H-Ala-[ The fluorescent conjugate 5 of (S)-RFAA(C8)]-Phe-OMe (diastereomer B) was obtained as a blue solid (54.3% yield calculated by fluorometer based on dye).
  • Peptide fluorescent conjugate 1 Alexa-Ala-Asp(C 8 F 17 )-Phe-NH 2 synthesized in Example 1
  • Peptide fluorescent conjugate 4 Alexa-Ala-[(R) -RFAA(C8)]-Phe-OMe
  • peptide fluorescent conjugate 5 Alexa-Ala-[(S)-RFAA(C8)]-Phe-OMe
  • the diethylamide form of the fluorescent substance Alexa Fluor647 fluorochrome 1
  • the DMSO solutions of synthesized peptide fluorescent conjugates 1, 4, 5, and fluorescent dye 1 were diluted with DMEM low-glucose culture medium to a concentration of 1.5 ⁇ M, and the DMSO concentration contained was 0.15%.
  • a sample solution for the cell membrane permeability test was prepared by adjusting the
  • HeLa cells were seeded in 96-well cell culture plates (0.5 ⁇ 10 5 cells/well) 24 hours before peptide treatment.
  • the medium of HeLa cells precultured at 37°C for 24 hours was replaced with sample solutions of peptide fluorescent conjugates 1, 4, 5 and fluorescent dye 1, and the cell membrane permeability was evaluated after 1 hour of incubation at 37°C.
  • the cell surface was washed three times with PBS (phosphate saline), and the cells were scraped off with Trypsin-EDTA 0.05% (manufactured by Gibco) and collected.
  • PBS phosphate saline
  • FIG. 1 shows the analysis results by flow cytometry of the cells cultured in the sample solution at 37° C. for 1 hour. The vertical axis is the number of cells (count), and the horizontal axis is the fluorescence intensity of each cell. Also, FIG. 2 shows the results of comparing the mean fluorescence intensity (MFI) of each sample.
  • the fluorescent peptide conjugate is indicated by PFCJ
  • the fluorescent dye is indicated by FD.
  • the fluorescence intensity of Alexa Fluor 647 in cells after incubation at 37°C for 1 hour showed Both were higher than cells treated with fluorescent dye 1.
  • the average fluorescence intensity of Alexa Fluor 647 in cells was significantly higher than that of cells treated with fluorochrome 1, compared to the peptide-fluorescent conjugates with fluoroalkyl groups attached directly to the ⁇ -carbon of the peptide backbone. 4 and the peptide fluorescent conjugate 5 treated with 4 and peptide fluorescent conjugate 5, whereas the peptide fluorescent conjugate in which a fluoroalkyl group was introduced to the carboxyl group of the side chain of aspartic acid via a peptide bond and a phenylene group.
  • Peptide fluorescent conjugate 1 Alexa-Ala-Asp(C 8 F 17 )-Phe-NH 2
  • Peptide fluorescent conjugate 2 Alexa-Ala-Asp(bis- C 4 F 9 )-Phe-NH 2
  • the diethylamide of the fluorescent substance Alexa Fluor 647 fluorescent dye 1
  • the commercially available cell membrane-permeable peptide Cys-TAT 47-57
  • a fluorescent substance, Alexa Fluor 647 C2 Maleimide was added to the terminal Cys (TAT-Alexa), and the uptake efficiency into cells was examined.
  • FIG. 3 shows the analysis results of cells cultured at 37° C. for 1 hour in the sample solution by flow cytometry.
  • FIG. 4 shows the results of comparing the mean fluorescence intensity (MFI) of each sample.
  • MFI mean fluorescence intensity
  • the fluorescence intensity of 647 was higher in cells treated with peptide fluorescent conjugates 1 and 2, in which a fluoroalkyl group was linked to the side chain via a linking group, than in cells treated with fluorescent dye 1 or TAT-Alexa. rice field.
  • the average fluorescence intensity of Alexa Fluor 647 in cells treated with TAT-Alexa was about twice that in cells treated with fluorescent dye 1. It was found that cells treated with peptide-fluorescent conjugates 1 and 2 were tens to hundreds-fold higher.
  • Peptide fluorescent conjugate 1 (Alexa-Ala-Asp(C 8 F 17 )-Phe-NH 2 ) containing a fluorine atom in the side chain synthesized in Example 1, Synthesized in Comparative Example 1 containing a fluorine atom in the side chain Efficiency of uptake into cells was examined for peptide fluorescent conjugate 3 (Alexa-Ala-Asp(C 12 H 25 )-Phe-NH 2 ) without peptide and fluorescent dye 1.
  • FBS fetal bovine serum
  • FIG. 5 shows the results of comparing the mean fluorescence intensity (MFI) of each sample with respect to the analysis results by flow cytometry of the cells cultured in the sample solution at 37° C. for 1 hour.
  • MFI mean fluorescence intensity
  • FIG. 6 shows the results of comparing the mean fluorescence intensity (MFI) of each sample with respect to the analysis results by flow cytometry of cells cultured at 37° C. for 1 hour in a sample solution containing FBS.
  • MFI mean fluorescence intensity
  • the mean fluorescence intensity (MFI) of Alexa Fluor 647 in cells after incubation for 1 hour at 37° C. was as shown in FIG. It was higher in cells treated with peptide fluorescent conjugate 3, which does not contain a fluorine atom in the side chain, than in cells treated with peptide fluorescent conjugate 1.
  • MFI mean fluorescence intensity
  • FIG. 7 shows the results of flow cytometry analysis of cells cultured in the sample solution at 37° C. for 1 hour.
  • FIG. 8 shows the result of comparing the relative fluorescence intensity (RFI) of each sample with the fluorescent dye 1 as a reference.
  • the peptide fluorescent conjugate is indicated by PFCJ
  • the fluorescent dye is indicated by FD.
  • FIG. 9 shows the analysis results of the average particle size (average size of number-based distribution) of each peptide fluorescent conjugate.
  • the peptide fluorescent conjugate is indicated by PFCJ, and the fluorescent dye is indicated by FD.
  • FIG. 10 shows the results of flow cytometry analysis of cells cultured in the sample solution at 37° C. for 1 hour.
  • FIG. 11 shows the results of comparing the mean fluorescence intensity (MFI) of each sample.
  • MFI mean fluorescence intensity
  • FIG. 12 shows the results of comparing the mean fluorescence intensity (MFI) of each sample.
  • MFI mean fluorescence intensity
  • Alexa Fluor of cells after incubation at 37°C for 1 hour.
  • the fluorescence intensity of 647 was higher in cells treated with peptide fluorescent conjugates 1 or 12, which had a fluoroalkyl group attached to the side chain via a linking group, than in cells treated with fluorochrome 1.
  • the average fluorescence intensity of Alexa Fluor 647 in cells treated with peptide fluorescent conjugate 12 was about 14 times higher than in cells treated with fluorochrome 1 . From these results, it was found that peptides with fluoroalkyl groups linked to side chains via linking groups have higher intracellular uptake efficiency and superior cell membrane permeability compared to fluorescent dyes.
  • the present invention provides peptides having amino acid residues containing fluorine atoms in side chains. Since the fluorine-containing peptide according to the present invention has excellent cell membrane permeability, it is expected to be used in the medical field as a physiologically active substance such as a carrier for introducing a medicinal ingredient into target cells.

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Abstract

La présente invention concerne un peptide ayant un groupe fluoroalkyle en tant que chaîne latérale. La présente invention concerne un peptide où deux ou plusieurs acides aminés sont liés à un peptide, sachant qu'au moins un des résidus d'acides aminés constituant le peptide possède une chaîne latérale de formule générale (1) [où : Z1 représente un groupe de liaison autre qu'un groupe alkylène di-, tri- ou tétra-valent ; Rf représente un groupe alkyle C1-30 substitué par au moins deux atomes de fluor, -SF5 ou -SF4-CR101R102-CR103R104Cl (où R101, R102, R103 et R104 représentent indépendamment un atome d'hydrogène, un atome de fluor ou un atome de chlore, à condition qu'au moins deux des R101, R102, R103 et R104 soient des atomes de fluor) ; n3 est 1, 2 ou 3 ; et le cercle noir représente une liaison].
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WO2008089491A2 (fr) 2007-01-19 2008-07-24 Kai Pharmaceuticals, Inc. Modifications de compositions peptidiques pour augmenter la stabilité et l'efficacité d'administration
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WO2008089491A2 (fr) 2007-01-19 2008-07-24 Kai Pharmaceuticals, Inc. Modifications de compositions peptidiques pour augmenter la stabilité et l'efficacité d'administration
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